The invention concerns certain mutant E. coli strains, and their use for performing processes for producing recombinant polypeptides.
Genomic study of higher organisms, micro-organisms, and viruses almost invariably requires, in addition to the cloning of their genes, large-scale production of their products (proteins), so as for example to obtain antibodies or to perform biochemical or crystallographic studies.
From the applications viewpoint, the utilization in the medical field of numerous human peptides and proteins also requires expression of corresponding genes in heterologous organisms.
Although expression systems have been established in various eukaryotic hosts (especially in yeasts, insects and primate cells), the most widely used host for these expression strategies remains the bacteria Escherichia coli (E. coli). The list of proteins of biotechnological or pharmacological interest that are produced in E. coli is extensive; classic examples include human insulin and human growth hormone.
The most well-known expression system in, prokaryotes was developed in the USA by the Studier and Richardson groups, during the 1980""s (Tabor and Richardson, 1985; Studier and Moffat, 1986). It is based on exploiting the properties of T7 RNA polymerase (namely RNA polymerase encoded by the T7 bacteriophage). That enzyme, which can be expressed in E. coli cells without toxicity, recognizes a very specific promoter. Any gene of interest (target gene) may be transcribed very efficiently, upon placing it downstream of this promoter and introducing it into an E. coli cell expressing T7 polymerase.
Nevertheless, in terms of expression, the results remain uncertain. Some target genes may be duly overexpressed, whereas others are expressed only moderately or not at all.
Previous work by the inventors revealed that one of the principal causes of these setbacks resides in the specific instability of the m-RNA synthesized by T7 RNA polymerase, which causes a decrease in the number of polypeptides synthesized by messaging (Lopez et al., 1994; Iost and Dreyfus, 1994, 1995). This instability is the consequence of the high speed of elongation of T7 RNA polymerase (Makarova et al., 1995). Specifically, the elongation speed of T7 polymerase, in contrast to that of bacterial RNA polymerase, is much greater than the translation speed of m-RNA by ribosomes. Nascent m-RNA is therefore exposed over most of its length, and is therefore readily attacked by nucleases, and in E. coli especially by the E-type ribonuclease (or RNase E), whose amino acid sequence is described by Casaregola et al. (Casaregola et al., 1992, 1994).
RNase E is an essential enzyme of E. coli; it is involved both in the degradation of m-RNA as well as in the maturation of ribosomal RNA (r-RNA). Mutations in the catalytic region (that is, in the N-terminal portion of RNase E) affect these two functions at the same time, and slow down or even arrest the growth of E. coli (Cohen and McDowall, 1997).
On the other hand, deletions in the C-terminal portion of RNase E do not affect the viability of E. coli. Specifically, by researching revertants of mutations in a protein (MukB) necessary for the segregation of chromosomes after replication, Kido et al. obtained various viable mutations in the rne gene, coding RNase E in E. coli, which cause synthesis of an RNase E that is truncated in its C-terminal portion (Kido et al., 1996). These authors concluded from these experiments that the C-terminal portion of RNase E is not essential for viability of E coli. They moreover formed the hypothesis that suppression of the mukB mutations by truncating of the RNase E, reflects the fact that truncated RNase E is less effective than the wild-type enzyme for degrading mukB m-RNA. Thus stabilized, a stronger synthesis of the mutant MukB protein could be achieved, thereby correcting the phenotype associated with the mutation. However, this stabilization of the mukB messenger was not demonstrated, and other authors proposed an entirely different interpretation to explain the suppressive effect of the truncating of RNase E on mukB mutations (Cohen and McDowall, 1997). These authors postulate in particular a direct interaction between RNase E and MukB. The basis for that idea is the fact that RNase E has a very substantial similarity with eukaryotic myosin (Casaregola et al., 1992: McDowall et al., 1993), which suggests that aside from its own RNase activity, it could, like MukB, play a structural role.
The present invention arises from the demonstration by the inventors of the fact that the truncating of RNase E causes an overall stabilization of cellular m-RNA, considered as a whole, as well as of the majority of individual m-RNAs that were examined, without significantly impeding the maturation of the r-RNAs (Lopez et al., 1999).
In that regard, the effect of the deletion is very different from that of a mutation in the N-terminal region, such as the ams mutation (Ono and Kuwano, 1979), renamed rne1 (Babitzke and Kushner, 1991), which confers thermosensitive activity to RNase E. For example, at 37xc2x0 C., this latter mutation causes a moderate increase in the lifespan of the m-RNAs (1.5 times each on average; the lifespan of the m-RNAs is here defined as the time during which they serve as a matrix for protein synthesis (Mudd et al., 1990a)), but it also causes a significant slowdown in maturation of the r-RNAs (estimated by the xe2x80x9cNorthernxe2x80x9d method; see Lopez et al., 1994) and it retards the growth by a factor of 2. On the contrary, deletion of the C-terminal portion of RNase E, especially of amino acids 586 to 1061 of this latter, causes a more significant stabilization of the m-RNA (two times on average), without causing a slowdown in the maturation of the r-RNA and without retarding growth. Thus, in hindsight, it is likely that the lack of growth that was observed with N-terminal mutations of RNase E, is due solely to the inability of the cells to mature r-RNA.
In summary, deletions in the C-terminal portion of RNase E have no effect on the activity of the catalytic domain, judging from the rapid maturation of the r-RNA. That rapid maturation explains why the cells containing such a deletion are viable. On the other hand, the deletion stabilizes the m-RNA as a whole, perhaps because it inhibits the association of the RNase E with other enzymes within a multi-protein structure, the so-called xe2x80x9cdegradosomexe2x80x9d, which might be necessary for degradation of the m-RNA (Carpousis et al., 1994; Miczack et al, 1996; Py et al., 1996; Kido et al., 1996; Cohen and McDowall, 1997). The important point from the perspective of the invention is that, by virtue of these deletions, it is possible to obtain E. coli strains having enhanced m-RNA stability, while preserving normal growth.
The inventors have also shown that the stabilization of m-RNA due to the deletion of the C-terminal portion of RNase E, is not uniform, but rather is more pronounced for less stable m-RNA. As is known, this is often the case for the m-RNA of xe2x80x9ctargetxe2x80x9d genes in expression systems. The contribution of this m-RNA to the overall protein synthesis is therefore enhanced by the presence of the deletion. E. coli strains comprising such a deletion therefore express recombinant exogenous polypeptides with sharply higher yields (in particular about 3 to 25 times higher) with respect to the expression yields of those recombinant polypeptides by E. Coli strains not comprising that mutation, especially when the expression of the said recombinant polypeptides is placed under the control of T7 RNA polymerase.
The present invention therefore has as an object to provide novel processes for producing recombinant proteins or polypeptides from E. coli, especially those of pharmaceutical or biological interest, at production yields substantially greater than those of the processes described up to now.
The present invention also has as an object to provide novel E. coli strains for practicing the above-mentioned processes, as well as methods for preparing such strains.
The present invention has as an object the use of E. coli strains whose gene encoding RNase E comprises a mutation such that the. enzyme produced upon expression of this mutated gene no longer possesses m-RNA-degrading activity, this mutation not significantly affecting the growth of the said E. coli strains, for practicing a process for producing predetermined exogenous recombinant polypeptides (or proteins).
The present invention more particularly concerns the use of E. coli strains whose gene coding RNase E comprises a mutation such that the enzyme produced upon expression of this mutated gene preserves the maturation activity of the r-RNA of the RNase E, but no longer possesses the degradation activity of the m-RNA, for practicing a process for producing predetermined exogenous recombinant polypeptides (or proteins).
The invention more particularly has as an object the above-mentioned utilization of E. coli strains as defined above, characterized in that the mutation consists in the substitution or deletion of one or several nucleotides in a region of the gene coding for the C-terminal portion of RNase E.
The invention yet more particularly concerns the above-mentioned utilization of E. coli strains as defined above, characterized in that the mutation corresponds to the substitution or to the deletion of one or several nucleotides of the region delimited by the nucleotide situated at position 1935 and the nucleotide situated at position 3623 of the DNA coding RNase E, represented by SEQ ID NO: 1.
Advantageously, the above-mentioned mutation causes modification or deletion of at least one amino acid from the C-terminal portion of RNase E.
To that end, the invention has as an object the above-mentioned utilization of E. coli strains as defined above, characterized in that the mutation causes the deletion of at least one, and up to all, of the last 563 amino acids of the sequence of RNase E represented by SEQ ID NO:2.
The invention more particularly has as an object the above-mentioned utilization of E. coli strains as defined above, characterized in that the mutation corresponds to the substitution of the guanine G in position 2196 of SEQ ID NO: 1 by a thymidine T, so as to create a stop codon TAA situated at positions 2196 to 2198 of SEQ ID NO:.
Advantageously, the above-mentioned mutant E. coli strains, used in the context of the invention, contain an exogenous inducible expression system, under the control of which is placed the expression of predetermined recombinant polypeptides, especially the inducible expression system using RNA polymerase of the T7 bacteriophage.
The invention also concerns E. coli strains that are transformed such that they contain an exogenous inducible expression system, and whose gene coding RNase E comprises a mutation such that the enzyme produced upon expression of this mutated gene no longer possesses degradation activity for m-RNA, this mutation not significantly affecting growth of the said E. coli strains.
The invention also has for an object E. coli strains such as described above, transformed such that they contain an exogenous inducible expression system, notably chosen from those described above, and whose gene coding RNase E comprises a mutation such that the enzyme produced upon expression of this mutated gene preserves the maturation activity for the r-RNA of the RNase E, but no longer possesses the activity of this latter for degradation of m-RNA.
The invention more particularly has as an object E. coli strains as described above, characterized in that the inducible expression system uses RNA polymerase coded by the T7 bacteriophage.
The invention also concerns E. coli strains as described above, characterized in that the mutation consists in the substitution or deletion of one or several nucleotides from the region of the gene coding for the C-terminal portion of RNase E.
The invention yet more particularly concerns E. coli strains as defined above, characterized in that the mutation corresponds to the substitution or deletion of one or several nucleotides from the region delimited by the nucleotide situated at position 1935 and the nucleotide situated at position 3623 of the DNA sequence coding RNase E, represented by SEQ ID NO: 1.
The invention more particularly has for an object mutant E. coli strains as defined above, characterized in that the above-mentioned mutation causes modification or deletion of at least one amino acid of the C-terminal portion of the RNase E expressed by the said strains.
To that end, the invention has as an object E. coli strains as defined above, characterized in that the mutation causes deletion of at least one, up to all, of the last 563 amino acids of the sequence of RNase E represented by SEQ ID NO: 2.
The invention more particularly has as an object E. coli strains as defined above, characterized in that the mutation corresponds to the substitution of guanine G at position 2196 of SEQ ID NO: 1, by thymidine T, so as to create a stop codon TAA situated at positions 2196 to 2198 of SEQ ID NO: 1.
The invention also has as an object E. coli strains as defined above, characterized in that the inducible expression system controls the transcription of a DNA sequence coding one or several predetermined recombinant polypeptides.
The invention also concerns any process for producing predetermined recombinant polypeptides, characterized in that it comprises:
a step of transforming E. coli strains whose gene coding RNase E comprises a mutation such that the enzyme produced upon expression of this mutated gene no longer possesses degradation activity for m-RNA, this mutation not significantly affecting the growth of the said E. coli strains, with a vector, especially a plasmid, containing the nucleotide sequence coding one or several recombinant polypeptides,
culturing of the transformed E. coli strains obtained during the preceding step, for a time sufficient to allow expression of the recombinant polypeptides in the E. coli cells,
and recovery of the recombinant polypeptide or polypeptides produced during the preceding step, if desired after purification of these latter, especially by chromatography, electrophoresis, or selective precipitation.
The invention more particularly has as an object any process for producing predetermined recombinant polypeptides, as defined above, characterized in that it comprises:
a step of transforming E. coli strains as described above, with a vector, especially a plasmid, containing the nucleotide sequence coding one or several recombinant polypeptides, so as to obtain the above-mentioned E. coli strains, in which transcription of the said nucleotide sequence coding one or several recombinant polypeptides is placed under the control of an inducible expression system,
culturing the transformed E. coli strains obtained during the preceding step, and inducing the said expression system, for a time sufficient to permit expression of the recombinant polypeptide or polypeptides in E. coli cells, the inducing of the said expression system especially being effected by causing synthesis of T7 RNA polymerase when the said expression system calls for that polymerase; this synthesis may notably be provoked by adding IPTG to the culture medium, or by raising the temperature, following which the gene coding for this RNA polymerase is placed under the control of a promoter regulated by the lac repressor (Studier and Moffat, 1986), or under the control of a thermo-inducible promoter (Tabor and Richardson, 1985),
and recovering the recombinant polypeptide or polypeptides produced during the preceding step.
A general process for obtaining mutant E. coli strains as described above, and capable of being used in the context of the present invention, comprises the following steps:
preparation of a plasmid containing an rne gene comprising a mutation as described above, and in which the promoter of the said rne gene is suppressed,
introduction of the plasmid obtained in the preceding step, into an E. coli strain comprising an inducible expression system, as well as a chromosomal mutation in the rne gene conferring a particular property to the said E. coli, such that the so-called rne1 mutation (Ono; and Kuwano, 1979) rendering the growth of the host thermosensitive, and permitting selecting acquisition of the desired mutation of the rne gene on the E. coli chromosome,
culturing the thus-transformed E. coli strains, and selecting E. coli strains having the particular property mentioned above, namely the clones resulting from the homologous recombination which permits replacing the said chromosomal mutation by the homologous sequence corresponding to the mutated rne gene of the said plasmid, especially selection of thermoresistant clones in the case where the chromosome mutation is the said rne1 mutation,
elimination of the plasmid from the selected clones, and identification from among these clones of those comprising the above-mentioned mutated rne gene, especially by analyzing by electrophoresis the length of the truncated RNase E polypeptide, coded by the above-mentioned mutated rne gene, produced by the mutant E. coli cells.